ABSTRACT Little is known regarding the physiochemical characteristics or health-related effects for the large array of aerosols that are inhaled by electronic cigarette (e-cig) users. We have therefore assembled a multidisciplinary team that combines expertise in aerosol sciences, in vitro high throughput screening (HTS), live animal imaging, and inhalation toxicology in order to address these issues. Our primary objective is to un- derstand how atomizer parameters and key ingredients in e-liquid, which are shared across different e-cig brands, contribute to undesirable aerosol characteristics and pulmonary toxicity. Based on our in vitro and in vivo preliminary data, the working hypotheses are (1) the heating coil temperature, chemical composition and vapor pressure of the major ingredients in e-liquid can be linked in a predictive manner to e-cig aerosol charac- teristics associated with toxicity; (2) a simulated lung exposure model employing air-liquid interface will provide novel insight into the dilution and dispersion of aerosol components and their potential for epithelial toxicity; and (3) correlations between HTS, aerosol composition, and endpoint toxicity measures will be validated when animals are exposed to e-cig aerosols in vivo. Three specific aims are proposed: (1) To systematically vary e- cig device parameters and e-liquid compositions to determine their impacts on aerosol physiochemical charac- teristics and in vitro toxicity; (2) To expose airway epithelial cells to e-cig aerosols as would occur in the human lung during vaping and determine toxicity signatures resulting from specific physiochemical features.; and (3) To determine acute and sub-chronic lung toxicity profiles resulting from exposures to e-cig aerosols us-ing transgenic animals. There are several novel aspects of this proposal that include: (1) a focus on key e-cig de- sign features (coil composition, coil resistance, and applied voltage) and e-liquid components (propylene glycol (PG), vegetable glycerin (VG), nicotine, and flavoring), that are shared across a wide range of brands, with a goal of developing predictive assessments linking their impact on aerosol characteristics to specific toxicity; (2) use of a mechanism-based HTS to systematically screen a large number of e-cig aerosols and identify those with toxicity signals that warrant further investigation; (3) development of a ventilated artificial lung model em- ploying primary human bronchial epithelial cells (HBEC)/air-liquid interface (ALI) cultures to carry out a detailed assessment of human exposure and detect early signals of epithelial toxicity; and (4) focused validation studies using transgenic mice and live animal imaging to assess the toxicity of acute and repeated exposure on the lung in vivo. This proposed research is significant in that it will identify key e-cig design parameters and e-liquid ingredients that can be readily assessed by HTS yet are linked in a predictive manner to more detailed measures of toxicity using human artificial lung and in vivo animal exposures. In addition, it is expected that these correlative outcomes and e-cig features, which are common to most brands, will facilitate the develop- ment of regulatory guidelines and e-cig design standards.